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PLoS One
2010 Apr 08;54:e10090. doi: 10.1371/journal.pone.0010090.
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An odorant receptor from the southern house mosquito Culex pipiens quinquefasciatus sensitive to oviposition attractants.
Pelletier J
,
Hughes DT
,
Luetje CW
,
Leal WS
.
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Insect odorant receptors (ORs) are heteromers comprised of highly variable odorant-binding subunits associated with one conserved co-receptor. They are potential molecular targets for the development of novel mosquito attractants and repellents. ORs have been identified in the malaria mosquito, Anopheles gambiae, and in the yellow fever mosquito, Aedes aegypti. However, they are still unknown in the Southern house mosquito, Culex quinquefasciatus, which transmits pathogens that cause human diseases throughout the world, including West Nile Virus in the United States. We have employed a combination of bioinformatics, molecular cloning and electrophysiology approaches to identify and characterize the response profile of an OR in Cx. quinquefasciatus. First, we have unveiled a large multigenic family of one-hundred-fifty-eight putative ORs in this species, including a subgroup of conserved ORs in three mosquito species. Using the Xenopus oocytes expression system, we have determined the response profile of CquiOR2, an antennae-specific OR, which shares high identity with putative orthologs in Anopheles gambiae (AgamOR2) and Aedes aegypti (AaegOR2). We show that CquiOR2 is highly sensitive to indole, an oviposition attractant for Cx. quinquefasciatus. The response profile of CquiOR2 expressed in Xenopus oocytes resembles that of an olfactory receptor neuron housed in the antennal short blunt-tipped sensilla (A2) of Cx. quinquefasciatus, which are natural detectors for oviposition attractants. This first Culex OR de-orphanized is, therefore, a potential molecular target for screening oviposition attractants.
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Figure 1. Phylogenetic relationships of mosquito ORs.Culex ORs are in black, Anopheles ORs are in blue and Aedes ORs are in red. Filled circles and empty circles represent 94â100% and 79â93% bootstrap support, respectively. The green box represents relationships in the conserved OR2-OR10 subgroup. CquiOR2 corresponds to (XM_001864509/XP_001864544), CquiOR9 to (XM_001864508/XP_001864543) and CquiOR10 to (XM_001844036/XP_001844088). Major species-specific expansions and conserved OR7 and OR8 subgroups are indicated.
Figure 2. Alignment of mosquito OR2 and OR10 amino acid sequences.Dark grey and light grey shading indicate residues conserved among three species and between two of the three species, respectively.
Figure 3. Expression profiles of two ORs in Cx. quinquefasciatus female tissues by RT-PCR.Olfactory tissues: antennae (FA); maxillary palps (FMp); proboscis (FPr). Non olfactory tissues: legs (FL); bodies (FB). CquiRpL8 was used as control gene.
Figure 4. CquiOR2 + CquiOR7 responds to indole, various methylindoles and 2-methylphenol.Xenopus oocytes expressing CquiOR2 + CquiOR7 were challenged with a panel of odorant compounds, each applied for 20 s at 10 µM. A) Upper left trace, an oocyte expressing CquiOR2 + CquiOR7 is challenged with 1-methylindole (1MI), 2-methylindole (2MI), 3-methylindole (3MI), 4-methylindole (4MI) and indole (IND). Upper right trace, an oocyte expressing CquiOR2 + CquiOR7 is challenged with 5-methylindole (5MI), 6-methylindole (6MI), 7-methylindole (7MI) and indole (IND). Lower left trace, an oocyte expressing CquiOR2 + CquiOR7 is challenged with phenol (Phe), 2-methylphenol (2MP), 3-methylphenol (3MP), 4-methylphenol (4MP), 4-ethylphenol (4EP), 2-butoxyethanol (2BE), geranylacetone (GA), 6-methyl-5-hepten-2-one (MHO), mosquito oviposition pheromone (MOP) and indole (IND). Lower right trace, an oocyte expressing CquiOR2 + CquiOR7 is challenged with octanal (OCT), nonanal (NON), decanal (DEC), 2-undecanone (2UD), 2-tridecanone (2TD), trimethylamine (TME) and indole (IND). B) Quantification of current responses of CquiOR2 + CquiOR7 expressing receptors. All responses are normalized to the response of the same oocyte to 10 µM indole (mean ± SEM, nâ=â3â4).
Figure 5. CquiOR2 + CquiOR7 is highly responsive to indole.A) Upper trace, an oocyte expressing CquiOR2 + CquiOR7 is challenged with 20 s applications of a range of concentrations of indole. Middle trace, an oocyte expressing CquiOR2 + CquiOR7 is challenged with 20 s applications of a range of concentrations of 3-methylindole. Lower trace, an oocyte expressing CquiOR2 + CquiOR7 is challenged with 20 s applications of a range of concentrations of 2-methylphenol. B) Concentration-response relationships for CquiOR2 + CquiOR7 expressing oocytes when activated with a range of indole, 3-methylindole and 2-methylphenol concentrations. All data are normalized to the response of each oocyte to 300 nM indole and the curves were fit as described in Materials and Methods (means ± SEM; nâ=â5â16). EC50 and nH values are provided in Table 1.
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